In Vivo Mapping of FACT-Histone Interactions Identifies a Role of Pob3 C-terminus in H2A-H2B Binding

Histone chaperones assist nucleosomal rearrangements to facilitate the passage of DNA and RNA polymerases through chromatin. The FACT (facilitates chromatin transcription) complex is a conserved histone chaperone involved in transcription, replication, and repair. The complex consists of two major subunits, Spt16 and SSRP1/Pob3 in mammals and yeast, which engage histones and DNA by multiple contacts. However, the precise mechanism of FACT function is largely unclear. Here, we used the genetically installed UV-activatable cross-linker amino acid p-benzoylphenylalanine (pBPA) to map the interaction network of FACT in living yeast. Unexpectedly, we found the acidic C-terminus of Pob3 forming cross-links to histone H2A and H2B most efficiently. This observation was independent of the performed cross-linking chemistry since similar histone cross-links were obtained using p-azidophenylalanine (pAzF). Further analyses identified a C-terminal nuclear localization sequence in Pob3. Its interaction with Importin-alpha interfered with H2A H2B binding, which suggests a possible regulatory role in FACT recruitment to chromatin. Deletion of acidic residues from the Pob3 C-terminus creates a hydroxyurea-sensitive phenotype in budding yeast, suggesting a potential role for this domain in DNA replication.